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Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
7
COMPARATIVE STUDY AMONG WATERMELON CRUD EXTRACT,
CITRULLINE AND LYCOPENE ON SOME REPRODUCTIVE INDICES
IN MALE MICE
Awatif Ibrahim Mohammed, Mohanad A. Al-Bayati;
BVM, MSc PhD. Pharmacology and Toxicology
aumnmumu@yahoo.com
Department of Pharmacology, College of the Veterinary Medicine, Baghdad University, Iraq.
Abstract
Objectives of this project were to study the effect of 70% crude alcoholic extract of watermelon Citrullus lanatus
pulp on the reproductive system of male mice as compared with two main component citrulline and lycopene. The
mice were divided into four groups, the first group were treated with watermelon 500mg/kg b.w., the second group
was treated with citrulline 30mg /kg b.w., third group was treated with lycopene 5mg/kg b.w. and treatment
continueous for 38 days while the fourth group used as a control group non treated. The study showed improvement
of most parameters related to increase the reproductive efficiency in alcoholic extract of watermelon and citrulline
treated group higher than as compared to lycopne andcontrol group.
Key word: watermelon, citrulline, lycopene, sperm parameter .
Introduction :
Nourishment has been linked with animal
reproductive success throughout history (1). Dietary
habits and essential nutrients to promote successful
reproductive out- comes have been identified for the
maternal peri-conceptional and peri-natal period, but
healthy dietary habits and essential nutrients for
paternal reproductive fitness are less clear (2).
Watermelon is a fruit of great economic importance,
with worldwide production Watermelon is enjoyed
by many people across the world as a fresh fruit,
partly owing to it being low in calories and highly
nutritious and thirst-quenching (3). Watermelon is
very rich in phytonutrients such as lycopene a
-carotene and a carotenoid of
excessive notice because of its antioxidant capacity in
scavenging reactive oxygen species, which cause
oxidative tissue destruction and loss of proper cell
function (4). Epidemiological revisions have
established that high consumption of fruits and
vegetables that high consumption of fruits and
vegetables containing lycopene is associated with
reduced incidence of coronary heart disease (5) and
some types of prostate and kidney cancer
furthermore, provoke sexual and reproductive
system. small intestine of rats (6) enables arginine to
be a non-essential amino acid by converting most
(83%) of citrulline to arginine in the kidney (7).
Citrulline-L-arginine is an essential amino acid that
has a wide role in reproductive, pulmonary, renal,
gastrointestinal, hepatic and immune systems and
expedites the healing of wounds (8). Citrulline is
used in the nitric oxide system in humans and
animals and has potential antioxidant and
vasodilatatory roles denoted to both of lycopene and
citrulline are promote events at cellular levels (9)
marvel production of nitric oxide and Carotenoids.
Now a days, peoples and animals condition are
concerned about their health and physical shape and
demand more natural ingredient foods of fresh
quality such as ready-to-eat, minimally processed or
fresh cut fruits and vegetables .
Materials and methods:
Watermelon extraction preparation:
The watermelon Citrullus lanatus was purchased
from the local market Baghdad and certified in the
Iraqi National Herbarium in Abougraib,16/9/2013.
The fresh watermelon washed with tabe water to
remove contaminante on rind (10) and decortications
with deseeding manually. The pulp segmented and
Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
8
blendering by electrical blender for 3minutes then
sieving with sieve at mesh 250 µm which were
forming slurry shape and saved in clean container
(11
Preparation of crude alcoholic extract (70%) and
dry of watermelon Citrullus lanatus pulp:
The powdered sample was weighed at 15g and the
extraction is employe by used of soxhlet apparatus
which contain two part electrical heater and magnetic
stirrer with a round glass flask which
dry powdered sample put into a thimble part then
extraction was clarified with magnatic sterring with
vacume pump machine.
4hrour (12&13). T
-
The body weight of males was
calculated as follow according to (14):
Body weight (gm) = b.w. after treatment b.w.before.
After 38 day of the treatment period finished, the
animals were anesthetized and the testis excised and
cleaned from the fat tissue. Testes weight and
compared to body weight as following : Testicular
weight. to body weight. ratio = Weight of testis (gm
)X 100/Weight of animal (gm). Then The volume of
testis was calculated by volumetric redual method
according to: Volume of testis = V2 volume of testis
after dip -V1 volume of testis before dip .
Epididymus Preparation:
epididymis were gently removed and freed from fat
tissue and immediately put in watch glass with 0.1ml
incise by micro scissor to 200 part (15)used by the
Spectrophotometric method to evaluate Lag time,
Velocity, fraction of rapidly moving sperm as
described by (16 and 17). Also Add 0.1ml of fresh
semen solutions on slide to evaluated sperm counting
after using of hemocytometer with using according to
(18): Sperm concentration= Number of sperms
1000 x4000/80. Also examining sperm abnormalities
by putting a drop of semen with drop of eosin and
negrosin stain on edge of slide examining with bright
field optics at ×100 according to (19) associated with
Acrosomal abnormal- lities according to (20) :
Acrosomal abnormalities % = number of counted
abnormal acrosome (%) / 200 x 100. Evaluation of
live and dead sperms were identified according to
(21): Live sperm%= Number of live sperms X100/
Total sperms number.
Animal groups:Total number of adult healthy male
mice (Swiss albino) 50 purchased from Laboratory
Animal Collage of Medicine University/ Baghdad,
weighted 22-34g, and aged 8 weeks, they were
housed
light schedule 12:12, with ventilated vaccum.
Mice were incubated in plastic cages (13x10x10) cm,
and cleaned daily, with substitute wood saw forth
time weekly. Mice left for adaptation period for 4
weeks.
Experimental design :
Group 1:Animals were served as control group given
orally distilled water for 38 days.
Group 2:Animals were received orally daily
watermelon alcoholic extract at dose 500mg /kg B.W
for 38 days (11 ).
Group 3:Animal were received orally daily Citrulline
at dose 30 mg/ kg B.W for 38 days (7). Group
4:Animal were received orally daily Lycopene at
dose 5 mg/kg B.W. for 38 days (22).
Results &descusions :
This study showed the watermelon treated group
display significant p<0.05 increase in body weight
and testicular morphometric parameter than both
citrulline and lycopene treated groupas in figure (1)
,table (1). In table 2 there were significant p<0.05
increase in sperm count in watermelon treated group
as compared to control group furthermore significant
p<0.05 higher than citrulline and lycopene treated
group. In table 3 there was significant p<0.05
increase in sperm count and viability & decrease in
sperm abnormalities in watermelon treated group as
compared to control group furthermore significant
p<0.05 higher than citrulline and lycopene treated
group. In table 4 there was significant p<0.05
increase in turdidimetric analysis of sperm motility in
watermelon treated group as compared to control
Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
9
group furthermore significant p<0.05 higher than
citrulline and lycopene treated group . That result
finding in table (1) may be due to ability of
watermelon to ameliorate and accelerate the
metabolic process by improved food intake (23) and
feed behaviors in animals under normal and stress
conditions (24) and had the ability to lowered the
subcutaneous adipose tissues and increase the brown
fat and muscle mass(25,26). In citrulline-L-arginine
considered as good appetizer(27)play important role
in stimulate the synthesis and releasing of growth
hormone (28), essential for body building by
reducing triglyceride in subcutaneous adipose tissue
and increasing muscle mass (29). That was clinically
showed as improvement in body weight (30)
&enhanced testicular parameter (31). While
Lycopene had little effect on body weight may be due
to the slight modulating in lean body mass with
reduction in fat tissue due to decrease the production
of cytokines, chimiokins and adipokines proteins
secreted by adipose tissue (32). Farther more
lycopene considered one of causal factor reduce
insulin levels which led to inhibition of Leptin was
adipose tissue products; led to reduction in hunger
with enhanced feelings of satiety (33). On other hand,
Sureda et al., (2010)(34) reported that watermelon-
citrulline-L-arginine-NO had the ability to enhanced
Insulin levels led to significant increased of growth
hormone secretion which considered as the major
growth metabolic regulatory hormone interact with
the receptor on the surface of cell membrane led to
activation of several signaling pathway and increase
the body weight or lean body mass(35) . That
increase in Growth Hormone was acts synergistically
with testosterone synthesis led to magnitude the
change in synthesis led to magnitude the change in
body mass as well as improvement the testicular
parameter that may (36) as in table (2). Watermelon-
citrulline-L-arginine had diverse metabolites
including ornithine, polyamines, proline, glutamate,
glutamite, creatinine, agrnatine, dimethylarginine
(37), spermidine and spermine (38). These metabolite
considered as good regulator for integrity and
function of cell membrane of sperm cell(39) led to
super increase in grow and count of sperm. That
mean there was positive correlations between
increase in citrulline-L-arginine concentration and
sperm concentration (40) as in (table 3). Also
watermelon had beneficial activation of the AMP-
activated protein kinase (41) led to provoke the rate
of glycolysis, resulting in higher rates of ATP
generation in spermatozoa (42) and consequently
induce formation of stabilizing disulfide cross-links
(43) led to increase chromatin compaction with DNA
arrangement (44) which considered important
determinant factor for sperm head morphology that
mean there was correlation between sperm DNA
stability and head morphology (45). That mean
watermelon and citrulline had the ability to increase
viability of sperm (46) and decrease any disorder in
sperm morphology. and that result agreed with
watermelon contain lycopene which showed that
increase in sperm count due to increase the
physiological activity of testicular cell associated
with reduction in testicular cell associated with
reduction in the apoptosis (47). In table (4)There was
significant p<0.05 decrease in watermelon and
citrulline derived treated group as compared to
compared to control group furthermore lesser than
lycopene treated group in lag time. Watermelon-
citrulline-L-arginine pathway may be increase sperm
concentration associated with increase in energy
utilization that cause low concentration of calcium
medium which may be effected the time lag required
for the initiated stimulation needed (48).
there was negative relationship between lag time and
sperm concentration (49). There was significant
p<0.05 increase in watermelon derived citrulline
treated group as compared to control group
furthermore watermelon derived citrulline more than
lycopene treated group in fraction
of rapid movement, velocity and motility index. In
watermelon-citrulline-L-argine there was increase in
sperm concentration and the motile will be the first
groups to swim upward into clear medium from a
concentrated cell suspension at the bottom mainly
dependent on time, increase turbidity of the medium
and increase in absorbance. The strongly motile
sperms may be the best candidates for fertilization
(50) and increase velocity of sperm trough enhance
the rate of glycolysis and higher rates of ATP
generation (42) and [Ca+2 ] increased the sperm
motility and velocity (51) as well as lycopene showed
the same increase increase in sperm motility and
velocity due to the antioxidant effect .
Conclusion:
The major aim of this thesis was to determine the
responsibility of nutrient on increase and/or
maximized the efficiency of male reproductive
function through via loading dose of alcoholic
extract of watermelon comparable with two main
active ingredient citrulline and lycopene or other
Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
10
constituted. In the last 38 days, this study was
developed and clarifies and provide a good indication
that administration of watermelon fruit which
considered as good source of different content e.g.
vitamin, Carbohydrate, trace salt beside the sufficient
amount of L-arginine-Nitric oxide which has agood
role in human health. So the endpoint of this study
was to reveal the major role of watermelon and two
main contain citrulline and lycopene on testicular
fitness and fertilized semen.
The major aim of this thesis was to determine the
endorse of watermelon nutrient capcitation and
maximized functional property of male reproductive
performance during loading dose of alcoholic extract
of watermelon in mice model, to ultimately design a
synergistic strategy for watermelon main active
ingredient citrulline, lycopene or other constituted
vitamin, Carbohydrate and trace salt. In the last 38
days, this study was developed and clarify supported
boosting idea. The watermelon administration and
citrulline-L-arginine-Nitric oxide pathway on
spermatogenesis and spermatogram and motility
indecies.
Characterize an upregulation of
spermatogenic cells yield in watermelon mice model
as a synergestic yield of lycopene and citrulline,
experiment joined and determined the sexual
hormone levels and their reflection on tissue
morphometric and stereological profile of peritubular
and seminiferous tubule testes. Finally watermelon
and their active ingredient treated group play a DNA
keeper and attendant in epididymis extracted
spermatozoa. That obviously marked reduction of
oxidative stress.
The endpoint increase fertility rate and refereed the
approach compatibility the watermelon derived
citrulline and lycopene on male productivity of
superior testicular fitness and fertilized semen.
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Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
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Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
12
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Wellbeing of the Whole Community. MNFC
Newsletter A Monthly Publication of the
Middlebury Natural Foods Cooperative.
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and Metabolism of Lycopene . Ealth sciences
center :1-133.
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(2008) Protective
effect of lycopene in cardio vascular disease
European Review for Medical and
Pharmacological Sciences.,12: 183-190.
48. S.,Osowska; C., Moinard; C., Loï; N., Neveux
and L., Cynober (2004) Citrulline increases
arginine pools and restores nitrogen balance after
massive intestinal resection. December; 53(12):
17811786.
49. Collins, J.K.; Wu, G.; Perkins-Veazie, P.; Spears,
K.; Claypool, P.L.; Baker, R.A. ; Clevidence,
B.A.)2007)Watermelon consumption increases
plasma arginine concentrations in adults.
Nutrition (burbank, los Angeles country,
califurnia) . Mar.,23(3):261-6.
Figure 1:The body weight changes parameter of male mice under loading doses of watermelon, citrulline and lycopene for 38
days.
Table 2:The testicular morphometrical in male mice under loading doses watermelon, citrulline and lycopene for 38 days.
Control
Watermelon
500mg/Kg.Bw
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Testicular weight g
0.020±0.001
0.058±0.216 A
0.053±0.017AB
0.030±0.008B
Testicular weight/body
weight ratio
0.077±0.020
1.895±0.060A 0.189±0.045B
0.113±0.040C
Testicular volume cm3
1.75±0.04089
3.16±2.116A 2.60±1.000B
2.50±0.489B
Testicular dimensions cm
0.018±0.10
0.307±0.3213A 0.272±0.3310 B
0.033±0.2102C
Testicular density g/cm3
0.0114±0.120
0.0183±1.264A 0.0203±0.216B
0.0120±0.155C
A B
C
0
2
4
6
8
body weight changes g
control watermelon citrulline lycopene
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
13
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 3: The Spermatogram parameter of male mice under loading doses of watermelon, citrulline and lycopene for 38 days.
Control
Watermelon
500mg/kgB.w
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Sperm concentration 106/ml
11.99±0.70
18.71±0.42
A
17.31±0.22
BA
16.66±0.97
B
Sperm viability%
85.77±1.36
94.18±1.52
A
91.02±1.46
BA
90.10±0.53
CB
Total spermabnormalities%
16.53±0.614
3.72±0.131
A
4.81±0.079
B
5.79±0.083
C
Primary%
7.05±0.58
0.61±0.101
A
0.43±0.30
B
0.27±0.22
C
Secondary%
9.37±0.872
2.50±0.045
A
3.99±0.072
B
4.08±0.011
B
Acrosome%
5.86±0.010
0.23±0.0028
A
0.17±0.0031
B
3.39±0.0047
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 4: The turbidimetric analysis of sperm motility parameter of male mice under loading doses of watermelon,
citrulline and lycopene for 38 days.
Control
Watermelon
500mg/kgB.w
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Lag time sec.
1.88±0.0062
0.69±0.0095
A
0.85±0.013
B
0.92±0.017
C
Fraction of rapidly moving
sperm
0.0174±0.0010
0.1557±0.0037
A
0.1408±0.0026
B
0.0819±0.0058
C
Velocity µm/sec
9.07±0.176
15.09±1.08
A
14.81±1.19
B
11.57±0.210
C
motility index %
1.20±0.048
2.97±0.067
A
2.44±0.039
B
2.00±0.761
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Figure 1:The body weight changes parameter of male mice under loading doses of watermelon, citrulline and lycopene for 38
days.
A B
C
0
2
4
6
8
body weight changes g
control watermelon citrulline lycopene
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Feb. 2014. Vol. 3, No.6 ISSN 2307-2083
International Journal of Research In Medical and Health Sciences
© 2013-2014 IJRMHS & K.A.J. All rights reserved
http://www.ijsk.org/ijrmhs.html
14
Table 2:The testicular morphometrical in male mice under loading doses watermelon, citrulline and lycopene for 38 days.
Control
Watermelon
500mg/Kg.Bw
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Testicular weight g
0.020±0.001
0.058±0.216 A
0.053±0.017AB
0.030±0.008B
Testicular weight/body
weight ratio
0.077±0.020
1.895±0.060A 0.189±0.045B
0.113±0.040C
Testicular volume cm3
1.75±0.04089
3.16±2.116A 2.60±1.000B
2.50±0.489B
Testicular dimensions cm
0.018±0.10
0.307±0.3213A 0.272±0.3310 B
0.033±0.2102C
Testicular density g/cm3
0.0114±0.120
0.0183±1.264A 0.0203±0.216B
0.0120±0.155C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 3: The Spermatogram parameter of male mice under loading doses of watermelon, citrulline and lycopene for 38 days.
Control
Watermelon
500mg/kgB.w
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Sperm concentration 106/ml
11.99±0.70
18.71±0.42
A
17.31±0.22
BA
16.66±0.97
B
Sperm viability%
85.77±1.36
94.18±1.52
A
91.02±1.46
BA
90.10±0.53
CB
Total spermabnormalities%
16.53±0.614
3.72±0.131
A
4.81±0.079
B
5.79±0.083
C
Primary%
7.05±0.58
0.61±0.101
A
0.43±0.30
B
0.27±0.22
C
Secondary%
9.37±0.872
2.50±0.045
A
3.99±0.072
B
4.08±0.011
B
Acrosome%
5.86±0.010
0.23±0.0028
A
0.17±0.0031
B
3.39±0.0047
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 4: The turbidimetric analysis of sperm motility parameter of male mice under loading doses of watermelon,
citrulline and lycopene for 38 days.
Control
Watermelon
500mg/kgB.w
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Lag time sec.
1.88±0.0062
0.69±0.0095
A
0.85±0.013
B
0.92±0.017
C
Fraction of rapidly moving
sperm
0.0174±0.0010
0.1557±0.0037
A
0.1408±0.0026
B
0.0819±0.0058
C
Velocity µm/sec
9.07±0.176
15.09±1.08
A
14.81±1.19
B
11.57±0.210
C
motility index %
1.20±0.048
2.97±0.067
A
2.44±0.039
B
2.00±0.761
C
Values are presented as mean ±SE